Dickinson C D, Shobe J, Ruf W
Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
J Mol Biol. 1998 Apr 10;277(4):959-71. doi: 10.1006/jmbi.1998.1639.
The catalytic activity of the trypsin-like serine protease coagulation factor VIIa is allosterically regulated. In this work, we employed monoclonal antibodies as probes to analyze conformational changes in the VII protease domain that are induced by zymogen activation, cofactor tissue factor (TF) binding, and active site occupancy. The epitopes of three monoclonal antibodies were mapped using a panel of 57 individual alanine replacement mutants in the protease domain. Two of the antibodies had typical "hot spot" epitopes in a basic cluster above the active site cleft and antibody binding to these epitopes was not affected by zymogen activation, TF binding, or active site occupancy. In contrast, the binding kinetics of VII/VIIa to a monoclonal antibody that mapped to an extended epitope overlapping with the macromolecular substrate exosite was affected by each of the conformational transitions of the VIIa protease domain. The changes in antibody affinity are consistent with a transition from zymogen VII to the TF.VIIa complex, with free enzyme VIIa as an intermediate that retains some zymogen-like features responsible for its low catalytic activity. In contrast, active site occupancy resulted in effects that were qualitatively different from the effects of zymogen activation on the antibody epitope. This provides novel insight into the conformational interdependence between the active site, the region for macromolecular substrate recognition, and the cofactor binding exosite of this allosterically regulated serine protease.
类胰蛋白酶丝氨酸蛋白酶凝血因子VIIa的催化活性受到别构调节。在本研究中，我们使用单克隆抗体作为探针，分析由酶原激活、辅因子组织因子（TF）结合和活性位点占据诱导的VII蛋白酶结构域的构象变化。使用蛋白酶结构域中的一组57个单个丙氨酸替代突变体对三种单克隆抗体的表位进行了定位。其中两种抗体在活性位点裂隙上方的碱性簇中有典型的“热点”表位，并且抗体与这些表位的结合不受酶原激活、TF结合或活性位点占据的影响。相比之下，VII/VIIa与一种单克隆抗体的结合动力学，该抗体的表位定位在与大分子底物外位点重叠的延伸表位上，受到VIIa蛋白酶结构域的每种构象转变的影响。抗体亲和力的变化与从酶原VII到TF.VIIa复合物的转变一致，游离酶VIIa作为中间体，保留了一些导致其低催化活性的类酶原特征。相比之下，活性位点的占据导致的效应在性质上与酶原激活对抗体表位的效应不同。这为这种别构调节的丝氨酸蛋白酶的活性位点、大分子底物识别区域和辅因子结合外位点之间的构象相互依赖性提供了新的见解。
